电穿孔调节白细胞介素-1受体阻滞剂基因抑制大鼠骨性关节炎
发布时间：2019-02-11 11:17
【摘要】：一、目的：基因治疗为骨性关节炎(Osteoarthritis, OA)的治疗提供了可能性,目前该疗法开展临床研究的瓶颈在于寻找一种安全有效的基因导入途径,本实验验证作为非病毒载体的电穿孔(Electroporation, EP)对骨性关节炎的治疗效果,并与研究最为广泛的腺相关病毒(Adeno-Associated Virus, AAV)载体作比较。二、方法：选用8周龄健康级雄性SD大鼠,在不同电穿孔参数下,使用电穿孔将荧光素酶(Luciferase)报告基因转染到大鼠膝关节组织内,采用小动物活体成像系统测定荧光强度,该强度反映在不同电穿孔参数下Luciferase基因表达水平,通过比较荧光强度以明确较佳的电穿孔参数。通过切断右膝前交叉韧带及切除内侧半月板前脚的方法建立骨性关节炎模型,在造模术后1周,按上述所选的较佳参数值进行下一步大鼠体内治疗实验。选择白细胞介素-1受体阻滞剂(Interleukin-1 receptor antagonist, IL-1Ra)作为目的基因,抑制由白细胞介素-1引起的炎症反应。根据不同的治疗方式将实验分为五组：单纯骨性关节炎未治疗组(OA组,n=24)：骨性关节炎单纯目的基因质粒治疗组(NP组,n=24);骨性关节炎电穿孔目的基因治疗组(EP组,n=24);骨性关节炎腺相关病毒转染目的基因治疗组(AAV组,n=24);正常对照组(Normal, n=24)。治疗后1周开始评价,每周取材1次,共计4次。取材前使用CatWalk进行步态分析,了解由于炎症引起的患肢疼痛情况;处死大鼠抽取右膝关节液进行ELISA检测,分析关节液中IL-1Ra、IL-1β、 TNF-a蛋白含量变化;分离右膝关节股骨远端,进行大体观观察,部分标本立即进行PCR检测,了解IL-1Ra、IL-1β、TNF-α、MMP-13、ADAMTS-4基因的表达情况,部分标本进行Micro-CT扫描以了解软骨下骨的重建情况,扫描后的样本进行固定脱钙、石蜡切片、病理染色,包括苏木素-伊红染色、番红O染色、甲苯胺蓝染色、IL-1Ra及Ⅱ型胶原的免疫组织化学染色。三、结果：在电穿孔参数脉宽为30ms,质粒量为50μg时,右侧膝关节表达的荧光强度明显高于其余各组(p0.05)。在EP组及AAV组,由炎症反应引起的疼痛水平明显的低于OA组和NP组(p0.05),于软骨缺损部位选取兴趣区,测得软骨下骨骨矿化密度、骨小梁厚度升高明显不及OA组和NP组(p0.05); PCR结果显示：EP组及AAV组IL-1Ra基因表达上调明显高于其余组(p0.05),前两周显示,AAV组的IL-1Ra表达量明显的高于EP组,后两周结果相反。在OA组和NP组，IL-1β、TNF-α表达明显上调。EP组及AAV组IL-1β、TNF-α表达高于正常对照但无统计学差异(p0.05)。MMP-13和ADAMTS-4的变化趋势类似于IL-1p和TNF-α、ELISA结果显示出与PCR相同的变化趋势;病理结果显示：4周时,EP组及AAV组未见明显软骨破坏及细胞外基质丢失,表达IL-1Ra阳性区域分布在软骨细胞核周围,表达Ⅱ型胶原阳性区域主要分布在软骨浅表层及中间层,两组间无差异。OA组和NP组软骨破坏严重,存留少许基质,局部区域呈现弱阳性IL-1Ra表达、弱阳性Ⅱ型胶原表达。四、结论：电穿孔即能获得与腺相关病毒相当的治疗效果又能较长时间的表达目的基因,将安全性考虑在内,电穿孔可以替代病毒载体用于骨性关节炎基因治疗的进一步实验研究。
[Abstract]:1. Objective: To provide the possibility of gene therapy for the treatment of Osteoarthritis (OA). At present, the bottleneck of this therapy is to find a safe and effective gene introduction route, and this experiment verifies the electroporation as a non-viral vector (Electrotrop, The effect of EP on the treatment of osteoarthritic arthritis is compared with the most widely used adeno-associated virus (AAV) vector. 2. The method comprises the following steps of: selecting 8-week-old healthy male SD rats, using electroporation to transfect a luciferase reporter gene into a rat knee joint tissue under different electroporation parameters, and measuring the fluorescence intensity by using a small-animal living body imaging system; This intensity reflects the expression level of the Lucifasse gene under different electroporation parameters, and by comparing the fluorescence intensity to define better electroporation parameters. A model of osteogenic arthritis was established by cutting the anterior cruciate ligament of the right knee and the anterior foot of the medial meniscus. Interleukin-1 receptor blocker (IL-1Ra) was selected as the target gene to inhibit the inflammatory response induced by interleukin-1. The experiment was divided into five groups according to different treatment methods: the non-treated group (OA group, n = 24), the pure-purpose gene plasmid treatment group (NP group, n = 24), and the gene therapy group (EP group, n = 24). Gene therapy group (AAV group, n = 24) for transfection of bone-related virus-related virus; normal control group (Normal, n = 24). The evaluation was started 1 week after treatment, and once a week, four times were obtained. Cavalk was used for gait analysis to understand the pain of the patient with limb pain due to inflammation, and the right knee fluid was extracted by ELISA, and the content of IL-1Ra, IL-1 and TNF-a in the joint fluid was analyzed, and the distal end of the right knee joint was isolated, and the general observation was performed. The expression of IL-1Ra, IL-1, TNF-1, MMP-13 and ADAMTS-4 was detected by PCR. The microCT scan of some specimens was performed to understand the reconstruction of the subchondral bone. The immunohistochemical staining of O-staining, toluidine blue staining, IL-1Ra and type II collagen were studied. The results showed that when the pulse width of the electroporation parameters was 30ms and the amount of the plasmid was 50. m u.g, the intensity of the expression of the right knee joint was significantly higher than that of the other groups (p0.05). In EP group and AAV group, the level of pain caused by inflammatory reaction was lower than that in OA group and NP group (p0.05). The expression of IL-1Ra in the EP group and the AAV group was significantly higher than that of the other group (p0.05). The expression of IL-1Ra in the AAV group was significantly higher than that of the EP group in the first two weeks. In the OA and NP groups, the expression of IL-1 and TNF-1 was up-regulated. The expression of IL-1 and TNF-1 in EP group and AAV group was higher than that of normal control, but there was no statistical difference (p0.05). The change trend of MMP-13 and ADAMTS-4 was similar to that of IL-1p and TNF-1, and the results of ELISA showed the same trend as that of PCR. In EP group and AAV group, no significant cartilage destruction and extracellular matrix loss were found, and the expression of IL-1Ra positive region was distributed around the nucleus of the cartilage. The expression of type 鈪，
本文编号：2419680
[Abstract]:1. Objective: To provide the possibility of gene therapy for the treatment of Osteoarthritis (OA). At present, the bottleneck of this therapy is to find a safe and effective gene introduction route, and this experiment verifies the electroporation as a non-viral vector (Electrotrop, The effect of EP on the treatment of osteoarthritic arthritis is compared with the most widely used adeno-associated virus (AAV) vector. 2. The method comprises the following steps of: selecting 8-week-old healthy male SD rats, using electroporation to transfect a luciferase reporter gene into a rat knee joint tissue under different electroporation parameters, and measuring the fluorescence intensity by using a small-animal living body imaging system; This intensity reflects the expression level of the Lucifasse gene under different electroporation parameters, and by comparing the fluorescence intensity to define better electroporation parameters. A model of osteogenic arthritis was established by cutting the anterior cruciate ligament of the right knee and the anterior foot of the medial meniscus. Interleukin-1 receptor blocker (IL-1Ra) was selected as the target gene to inhibit the inflammatory response induced by interleukin-1. The experiment was divided into five groups according to different treatment methods: the non-treated group (OA group, n = 24), the pure-purpose gene plasmid treatment group (NP group, n = 24), and the gene therapy group (EP group, n = 24). Gene therapy group (AAV group, n = 24) for transfection of bone-related virus-related virus; normal control group (Normal, n = 24). The evaluation was started 1 week after treatment, and once a week, four times were obtained. Cavalk was used for gait analysis to understand the pain of the patient with limb pain due to inflammation, and the right knee fluid was extracted by ELISA, and the content of IL-1Ra, IL-1 and TNF-a in the joint fluid was analyzed, and the distal end of the right knee joint was isolated, and the general observation was performed. The expression of IL-1Ra, IL-1, TNF-1, MMP-13 and ADAMTS-4 was detected by PCR. The microCT scan of some specimens was performed to understand the reconstruction of the subchondral bone. The immunohistochemical staining of O-staining, toluidine blue staining, IL-1Ra and type II collagen were studied. The results showed that when the pulse width of the electroporation parameters was 30ms and the amount of the plasmid was 50. m u.g, the intensity of the expression of the right knee joint was significantly higher than that of the other groups (p0.05). In EP group and AAV group, the level of pain caused by inflammatory reaction was lower than that in OA group and NP group (p0.05). The expression of IL-1Ra in the EP group and the AAV group was significantly higher than that of the other group (p0.05). The expression of IL-1Ra in the AAV group was significantly higher than that of the EP group in the first two weeks. In the OA and NP groups, the expression of IL-1 and TNF-1 was up-regulated. The expression of IL-1 and TNF-1 in EP group and AAV group was higher than that of normal control, but there was no statistical difference (p0.05). The change trend of MMP-13 and ADAMTS-4 was similar to that of IL-1p and TNF-1, and the results of ELISA showed the same trend as that of PCR. In EP group and AAV group, no significant cartilage destruction and extracellular matrix loss were found, and the expression of IL-1Ra positive region was distributed around the nucleus of the cartilage. The expression of type 鈪，
本文编号：2419680
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